Perhaps the most important aspect in diagnosing the cause of symptoms experienced by a patient when an infectious agent is suspected as being the cause of the symptoms is the establishment of the identity of the specific organism that is etiologically responsible for the symptoms. Additionally, a significant need exists for a method to identify people and animals that have been infected with an organism, such as a microorganism, including people and animals who are not exhibiting signs or symptoms of disease associated with the organism.
Classically, the presence of infection due to a particular infectious microorganism has been established by isolating the organism from the body of a patient, culturing the organism on a suitable culture medium, and identifying the cultured organism based on biochemical, immunological, or other tests. This method suffers from several disadvantages. Diagnosis by culture and identification often requires a substantial period of time when growing organisms that have a slow growth rate. For example, standard culture and identification methods for Mycobacterium avium subsp. paratuberculosis, the causative organism of Johne's disease in cattle and Crohn's disease in people, may require 8 to 16 weeks or more to perform due to the very slow growth rate of this organism. Another disadvantage to culture and identification methods of diagnosis is that the particular organism causing disease in a patient may fail to grow on standard culture media, leading to a negative culture result and a failure in diagnosis. Additionally, because such methods require the isolation of an infectious organism from a patient, these methods are inappropriate at times when the patient is not shedding the organism or if the organism is located in an inaccessible location within the body of the patient.
In recent years, molecular biological and immunological methods have been developed for the diagnosis of infectious diseases. These methods generally fall into three categories, detection of genome nucleic acids, detection of protein, and detection of antibodies directed against a pathogen.
Diagnosis by identification of genome nucleic acids is typically performed using either or both amplification of DNA by polymerase chain reaction (PCR) followed by identification of PCR fragments produced or by use of probes that bind specifically to a portion of the genome of a suspected causative organism. These methods, especially when used in combination, can be very sensitive and specific methods to establish a diagnosis of a causative organism. There are several disadvantages associated with these methods. They are expensive, require sophisticated technical expertise to perform, and generally take several days to obtain enough microorganisms for a diagnosis. Another significant disadvantage associated with diagnosis by detection of genome nucleic acids is that an organism must be isolated in order to obtain the genome nucleic acids.
Diagnosis by identification of proteins is typically performed by an enzyme-linked immunosorbent assay (ELISA). In this test, an antigen from a microorganism, typically a disrupted microorganism or a portion of a microorganism, is bound to a solid support and reacted with a first antibody in a test sample, typically serum, that is specific for the antigen of interest. A labeled second antibody that binds to antibodies in test serum is then exposed to the solid support complex to provide a means for identification of the presence of the antigen. ELISA tests, however, suffer from several disadvantages including low sensitivity and the requirement to provide two different antibodies for the detection of an antigen. ELISA testing requires skilled laboratory technicians and can provide false results if samples contain cross-reacting antibodies.
An example of an infectious disease for which currently available diagnostic methods are inadequate is Johne's disease, a disease in cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP). Johne's disease results in decreased milk production and early culling of infected cows resulting in an annual loss of approximately $1.5 billion to the agricultural industry in the United States. Considerable evidence exists that MAP is also the causative organism of Crohn's disease in humans. Despite this significant impact on the U.S. economy and on human health, there is no effective diagnostic test to determine early infections by MAP.
At present, fecal culture is considered to be the most accurate means of diagnosing MAP infection. However, this diagnostic test has low sensitivity (less than 50%) and is capable of detecting infections only in animals that are actively shedding MAP in their feces. Additionally, diagnosis of MAP by culture typically requires 8 to 16 weeks for growth of the organism.
Other diagnostic tests for Johne's disease include PCR, complement fixation, agar gel immunodiffusion, and ELISA. These tests, each of which utilizes a molecular extract of MAP, have inherently low specificity or sensitivity for MAP and suffer from the disadvantages present with these methods as indicated above.
A significant need exists for a diagnostic method to detect infection by an organism, such as a microorganism, that can be performed rapidly, is highly sensitive, is highly specific, and which may be performed by an individual lacking sophisticated laboratory training. Particularly, a significant need exists for such a diagnostic method that is useful for diagnosing diseases such as those caused by MAP.